WO1999011782A1 - T-cell membrane protein (tirc7), peptides and antibodies derived therefrom and uses thereof - Google Patents

T-cell membrane protein (tirc7), peptides and antibodies derived therefrom and uses thereof Download PDF

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Publication number
WO1999011782A1
WO1999011782A1 PCT/EP1998/005462 EP9805462W WO9911782A1 WO 1999011782 A1 WO1999011782 A1 WO 1999011782A1 EP 9805462 W EP9805462 W EP 9805462W WO 9911782 A1 WO9911782 A1 WO 9911782A1
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Prior art keywords
cell
tirc7
protein
antibody
membrane protein
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PCT/EP1998/005462
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English (en)
French (fr)
Inventor
Nalan Utku
Steven R. Gullans
Edgar L. Milford
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Brigham And Women's Hospital, Inc.
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Priority claimed from DE19738710A external-priority patent/DE19738710A1/de
Priority to EP98945293A priority Critical patent/EP0996726B1/de
Priority to JP2000508792A priority patent/JP4459435B2/ja
Priority to DE69820449T priority patent/DE69820449T2/de
Priority to AU92654/98A priority patent/AU751151C/en
Priority to DK98945293T priority patent/DK0996726T3/da
Application filed by Brigham And Women's Hospital, Inc. filed Critical Brigham And Women's Hospital, Inc.
Priority to AT98945293T priority patent/ATE256185T1/de
Priority to CA2301499A priority patent/CA2301499C/en
Publication of WO1999011782A1 publication Critical patent/WO1999011782A1/en
Priority to US11/126,866 priority patent/US7863418B2/en
Priority to US11/126,841 priority patent/US20050271659A1/en
Priority to US11/510,868 priority patent/US7931901B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • T-cell membrane protein TIRC7
  • the present invention pertains generally to a T cell immune response cDNA 7 (TIRC7) encoding a novel T-cell transmembrane protein as well as peptides und polypeptides derived therefrom and antibodies recognizing said (poly)peptides.
  • TIRC7 T cell immune response cDNA 7
  • the present invention relates to TIRC7 cDNA and its encoded protein.
  • the present invention relates to polynucleotides derived from said TIRC7 cDNA encoding a peptide or polypeptide being capable of inhibiting T-cell stimulation through the T-cell membrane protein (TIRC7).
  • the present invention relates to vectors comprising such polynucleotides and host cells transformed therewith as well as their use in the production of the above-defined peptides or polypeptides.
  • the present invention relates to the (poly)peptide encoded by said polynucleotides or obtainable by the method of the invention.
  • the present invention relates to antibodies against said peptides and polypeptides that are capable of inhibiting T-cell stimulation through the T-cell membrane protein (TIRC7).
  • the present invention additionally relates to pharmaceutical and diagnostic compositions comprising the aformentioned peptide, polypeptide, or antibody.
  • the present invention relates to methods and uses for modulating immune responses through the novel TIRC7 membrane protein as well as to pharmaceutical compositions comprising agents which act on the TIRC7 membrane protein or its ligand. Also, the invention relates to the use of the before-described polynucleotide, vector, peptide, polypeptide, or antibody for the preparation of pharmaceutical compositions for use in organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, or for treatment of tumors. Furthermore, the present invention relates to methods for modulating (antigen-specific) T cell unresponsiveness. The present invention encompasses methods for inducing, maintaining or reversing T cell unresponsiveness by inhibiting or stimulating an (unresponsive) T cell through the novel TIRC7 membrane protein.
  • T cell activation is a serial process involving multiple signaling pathways and sequential changes in gene expression resulting in differentiation of T cells into distinct subpopulations, i.e. Th1 and Th2, which are distinguishable by their pattern of cytokine production and characterize the mode of the cellular immune response (Abbas et al., 1996; Crabtree, 1989).
  • the T cell response is initiated by the interaction of the antigen- specific T cell receptor (TCR) with peptide presented by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • TCR antigen- specific T cell receptor
  • MHC major histocompatibility complex
  • Additional signals are provided by a network of receptor-ligand interactions mediated by a number of membrane proteins such as CD28/CTLA4 and B7, CD40/CD40L, LFA-1 and ICAM-1 (Lenschow et al., 1996; Linsley and Ledbetter, 1993; Xu et al., 1994, Bachmann et al., 1997; Schwartz, 1992), collectively called costimulatory signals (Perez et al., 1997).
  • membrane proteins can alter T cell activation in distinct ways (Bachmann et al., 1997) and regulate the immune response by the integration of positive and negative signals provided by these molecules (Bluestone, 1995; Perez et al., 1997).
  • the present invention relates to polynucleotides encoding a novel T-cell membrane protein. Furthermore, the present invention relates to peptides and polypeptides derived therefrom as well as to antibodies capable of inhibiting T-cell stimulation through the novel T-cell membrane protein. More particulary, the present invention relates to applications in the medical field that directly arise from the polynucleotides, peptides, (poly)peptides and antibodies of the invention. Additionally, the present invention relates to a novel method for testing activators and inhibitors of T-cell proliferation.
  • compositions, methods and uses of the invention are useful therapeutically in situations where it is desirable to modulate (antigen- specific) immune responses, e.g., inducing and maintain (antigen-specific) T-cell unresponsiveness or restore (antigen-specific) T-cell responsiveness.
  • T-cell unresponsiveness can be maintained by blocking TIRC7 stimulation in a subject who has an autoimmune disease to alleviate symptoms of the autoimmune disease.
  • a TIRC7 inhibitory agent is administered to the subject in an amount and over a period of time sufficient to maintain T-cell unresponsiveness.
  • T-cell unresponsiveness can be reversed in a subject bearing a tumor to stimulate a tumor specific T-cell response or in a subject receiving a vaccine to enhance the efficacy of the vaccine.
  • a cell e.g., a tumor cell
  • a TIRC7 stimulatory agent can be administered to the subject bearing a tumor or who has had a tumor surgically removed to prevent recurrence of the tumor.
  • antigen-specific responsiveness can be restored to anergized T-cells in vitro by stimulating the T- cells through TIRC7. Responsive T-cells generated in vitro can then be administered to a subject.
  • the technical problem of the invention is to provide means and methods for the modulation of T-cell responses which are particularly useful in organ transplantation and autoimmune diseases.
  • the solution to this technical problem is achieved by providing the embodiments characterized in the claims, namely a novel T-cell membrane protein encoded by T- cell Immune Response cDNA7 (TIRC7) is described which exhibits a central role in T-cell activation in vitro and in vivo.
  • TIRC7 mRNA is transiently upregulated in the early phase of T-cell activation via a calcineurin-dependent pathway.
  • Example 2 the TIRC7 protein encoding cDNA has been cloned and characterized; see the first part of Example 1 and Figure 1. Furthermore, said cDNA was subjected to in vitro translation and preliminary results obtained in MLR inhibition tests with the in vitro translated TIRC7 protein suggested a putative immunosuppressive potential of said protein, see Example 2 with Figure 5B.
  • a second (independent) set of experiments performed in accordance with the present invention (Examples 2 to 4), surprisingly revealed that modulation of TIRC7 membrane protein mediated signals with specific anti-TIRC7 membrane protein antibodies in vitro efficiently prevents T-cell proliferation and IL-2 secretion which is reversible by exogenous IL-2.
  • Anti-TIRC7 membrane protein antibodies specifically inhibit Th1 subset specific cytokine expression but spare the Th2 cytokines.
  • Administration of said antibodies in rats significantly prolongs kidney allograft survival.
  • the latter described results obtained in accordance with the present invention provide evidence for an essential role of TIRC7 membrane protein in the early events of T cell activation.
  • targeting of TIRC7 membrane protein and its encoding gene provides a novel therapeutic approach for modulation of the immune response.
  • the invention relates to a polynucleotide encoding a TIRC7 membrane protein or a biologically active fragment thereofcomprising a DNA sequence selected from the group consisting of (i) DNA sequences comprising a nucleotide sequence the amino acid sequence depicted in SEQ ID NO. 2 or SEQ ID NO. 13 from amino acid position 1 to
  • DNA sequences comprising a nucleotide sequence encoding a fragment or derivative of the protein encoded by the DNA sequence of (i) or (ii); and (iv) DNA sequences the complementary strand of which hybridizes with and which is at least 70% identical to the polynucleotide as defined in any one of (i) to
  • TIRC7 membrane protein denotes a protein involved in the signal transduction of T-cell activation and/or proliferation and that, preferably in a soluble form is capable of inhibiting or suppressing T-cell proliferation in response to alloactivation in a mixed lymphocyte culture or in response to mitogens when exogeneously added to the culture.
  • soluble in vitro translated TIRC7 protein is able to efficiently suppress in a dose dependent manner the proliferation of T-cells in response to alloactivation in a mixed lymphocyte culture or in response to mitogens; see Example 2, Figure 5B.
  • biologicalcally active fragment thereof refers to peptides and polypeptides that are derived from said TIRC7 membrane protein and that are capable of inhibiting T-cell proliferation as defined above.
  • a cDNA fragment was amplified by the Reverse Transcription Differential Display Polymerase Chain Reaction (DDRT-PCR) technique from RNA from cells stimulated in mixed lymphocyte culture (MLC).
  • DDRT-PCR Reverse Transcription Differential Display Polymerase Chain Reaction
  • the amino acid sequence of the 350 base-pair (bp) cDNA fragment thus obtained was reported to belong to a new human (H+)-ATPase proton pump homologue differentially expressed in alloactivated lymphocytes as the amino acid sequence had substantial homology to a rat and bovine vesicular (H+)-ATPase proton pump, respectively.
  • a polynucleotide with the nucleotide sequence of the coding region as depicted in SEQ ID NO: 1 has been identified encoding a protein of 614 amino acids (SEQ ID NO: 2) with a molecular weight of 75 kDA.
  • SEQ ID NO: 2 a protein of 614 amino acids
  • TIRC7 membrane protein is expressed in all lymphoid tissues with low expression only in thymus, bone marrow and fetal liver and is transiently up regulated in lymphocytes after stimulation of the T-cell receptor, see Example 1 , Figure 3.
  • the TIRC7 encoding gene has been located by using the fluorescence-in situ- hybridization (FISH) method on the long arm of human chromosome 10 (13.4-13.5q) which is close to the breakpoint region of the bcl-gene associated with leukemia.
  • FISH fluorescence-in situ- hybridization
  • the TIRC7 membrane protein is predominantly expressed on the cell membrane, consistent with a target for an external ligand; see Example 1 , Figure 4.
  • the seven transmembrane domain structure predicts three extracellular loops and an extracellularly oriented carboxy terminus; see Figure 2. From the above it is evident that the nucleotide sequence depicted in SEQ ID NO. 1 encodes a novel class of T-cell membrane proteins.
  • nucleotide sequence By the provision of this nucleotide sequence it is now possible to isolate identical or similar polynucleotides which code for proteins with the biological activity of TIRC7 from other species or organisms.
  • Well-established approaches for the identification and isolation of such related sequences are, for example, the isolation from genomic or cDNA libraries using the complete or part of the disclosed sequence as a probe or the amplification of corresponding polynucleotides by polymerase chain reaction using specific primers.
  • a further polynucleotide encoding a TIRC7 membrane protein was isolated using a nucleic acid molecule comprising the coding sequence of SEQ ID NO: 1 as a probe.
  • the nucleotide sequence of said polynucleotide is given in SEQ ID NO: 12 encoding a protein having the amino acid sequence of SEQ ID NO: 13.
  • the nucleotide and amino acid sequences of said TIRC7 membrane protein are substantially identical with those of the TIRC7 membrane protein encoded by SEQ ID NO: 1 except at amino acid position 121 (Arg ⁇ Gin) and, therefore, presumably represent allelic variants.
  • the invention also relates to polynucleotides which hybridize to the above described polynucleotides and differ at one or more positions in comparison to these as long as they encode a TIRC7 membrane protein as defined above.
  • Such molecules comprise those which are changed, for example, by deletion(s), insertion(s), alteration(s) or any other modification known in the art in comparison to the above described polynucleotides either alone or in combination.
  • Methods for introducing such modifications in the polynucleotides of the invention are well-known to the person skilled in the art; see, e.g., Sambrook et al. (Molecular cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)).
  • the invention also relates to polynucleotides the nucleotide sequence of which differs from the nucleotide sequence of any of the above-described polynucleotides due to the degeneracy of the genetic code.
  • hybridizing in this context is understood as referring to conventional hybridization conditions, preferably such as hybridization in
  • hybridizing refers to stringent hybridization conditions, for example such as described in Sambrook, supra.
  • polynucleotides which share 70%, preferably at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above-mentioned polynucleotides and have the same biological activity.
  • Such polynucleotides also comprise those which are altered, for example by nucleotide deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison to the above-described polynucleotides. Methods for introducing such modifications in the nucleotide sequence of the polynucleotide of the invention are well known to the person skilled in the art.
  • the present invention encompasses any polynucleotide that can be derived from the above described polynucleotides by way of genetic engineering and that encode upon expression a TIRC7 membrane protein or a biologically active fragment thereof.
  • regulatory sequences may be added to the polynucleotide of the invention.
  • promoters, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide of the invention may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
  • the invention relates to nucleic acid molecules of at least 15 nucleotides in length hybridizing specifically with a polynucleotide as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences encoding no or substantially different proteins. Such nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of polynucleotides according to the invention. Another application is the use as a hybridization probe to identify polynucleotides hybridizing to the polynucleotides of the invention by homology screening of genomic DNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a polynucleotide as described above may also be used for repression of expression of a gene comprising such a polynucleotide, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a polynucleotide of the invention.
  • nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
  • said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucieases in the cell.
  • Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
  • Such nucleic acid molecules may further contain ribozyme sequences as described above.
  • polynucleotide of the invention can be used for "gene targeting” and/or “gene replacement”, for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
  • nucleic acid molecules are labeled.
  • Methods for the detection of nucleic acids are well known in the art, e.g., Southern and northern blotting, PCR or primer extension.
  • said nucleic acid molecules may be used for the suppression of TIRC7 expression.
  • the polynucleotide of the invention encoding the above described TIRC7 membrane protein or biologically active fragments thereof may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • said polynucleotide is part of a vector.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the P L , lac, trp or tac promoter in E.
  • regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40- enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI , pcDNA3 (ln-vitrogene), or pSPORTI (GIBCO BRL).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the protein of the invention may follow; see, e.g., the appended examples.
  • the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention.
  • vectors particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention.
  • Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.
  • the present invention relates to a cell containing the polynucleotide or vector described above.
  • said cell is a eukaryotic, most preferably a mammalian cell if therapeutic uses of the protein are envisaged.
  • yeast and less preferred prokaryotic, e.g., bacterial cells may serve as well, in particular if the produced protein is used as a diagnostic means.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • prokaryotic is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a protein of the invention.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells.
  • the protein encoded by the polynucleotide of the present invention may be glycosylated or may be no ⁇ -glycosylated.
  • TIRC7 proteins of the invention may also include an initial methionine amino acid residue.
  • a polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructs and methods described therein can be utilized for expression of the TIRC7 protein of the invention in eukaryotic or prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • transgenic animals preferably mammals, comprising cells of the invention may be used for the large scale production of the TIRC7 protein of the invention.
  • an animal, preferably mammalian cell naturally having a polynucleotide of the invention present in its genome can be used and modified such that said cell expresses the endogenus gene corresponding to the polynucleotide of the invention under the control of an heterologous promoter.
  • the introduction of the heterologous promoter which does not naturally control the expression of the polynucleotide of the invention can be done according to standard methods, see supra. Suitable promoter include those mentioned hereinbefore.
  • the present invention relates to a method for the production of a TIRC7 membrane protein or a biologically active fragment thereof comprising:
  • the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the TIRC7 protein of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
  • the protein of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982).
  • Substantially pure proteins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses.
  • the proteins may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures.
  • the present invention relates to a TIRC7 membrane protein or a biologically active fragment thereof encoded by the polynucleotide of the invention or produced by a method of as described above.
  • the protein of the invention can be further coupled to other moieties as described above for, e.g., drug targeting and imaging applications. Such coupling may be conducted chemically after expression of the protein to site of attachment or the coupling product may be engineered into the protein of the invention at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.
  • the provision of the TIRC7 protein of the present invention enables the production of TIRC7 specific antibodies.
  • hybridoma technology enables production of cell lines secreting antibody to essentially any desired substance that produces an immune response.
  • RNA encoding the light and heavy chains of the immunoglobulin can then be obtained from the cytoplasm of the hybridoma.
  • the 5' end portion of the mRNA can be used to prepare cDNA to be inserted into an expression vector.
  • the DNA encoding the antibody or its immunoglobulin chains can subsequently be expressed in cells, preferably mammalian cells.
  • renaturation techniques may be required to attain proper conformation of the antibody.
  • point substitutions seeking to optimize binding may be made in the DNA using conventional cassette mutagenesis or other protein engineering methodology such as is disclosed herein.
  • the present invention also relates to an antibody specifically recognizing the peptide or polypeptide of the invention.
  • the present invention relates to a cell that has been modified to express a TIRC7 protein or an antibody of the invention.
  • This embodiment may be well suited for, e.g., restoring T-cell responsiveness to an antigen, in particular if the antibody of the invention capable of stimulating T-cell proliferation is expressed in a form suitable to be presented on the cell surface.
  • the present invention relates to pharmaceutical compositions comprising a peptide or polypeptide being capable of inhibiting T-cell stimulation through the TIRC7 membrane protein and/or being recognized by an antibody capable of inhibiting T-cell stimulation through the TIRC7 membrane protein encoded by a fragment of the above described polynucleotides or an antibody specifically recognizing said peptide or polypeptide.
  • the term "capable of inhibiting T-cell stimulation through TIRC7 membrane protein” denotes the ability of suppressing the proliferation of T-cells in response to alloactivation in a mixed lymphocyte culture or in response to mitogens by way of blocking or antagonizing the biological activity of the TIRC7 membrane protein herein also referred to as TIRC7 protein or TIRC7.
  • the terms "capable of inhibiting T-cell stimulation through TIRC7 membrane protein” and “inhibiting TIRC7 activity” are used interchangeable herein.
  • anti-TIRC7 antibodies directed against the extracellular domains are able to efficiently suppress the proliferation of T-cells in response to alloactivation in a mixed lymphocyte culture or in response to mitogens; see Example 2, Figure 5. Similar results were obtained with in vitro translated soluble TIRC7 protein.
  • the inhibitory effect of anti-TIRC7 antibodies on T-celis induced by a variety of different stimulatory pathways suggests that TIRC7 plays a central role in T cell activation.
  • inhibition of T cell proliferation in MLR by antibody targeting of TIRC7 suggest the existence of a ligand specifically interacting with TIRC7.
  • Antibody blocking of costimulatory molecules has been shown to inhibit human T cell proliferation (Linsley et al., 1992; Walunas et al., 1994). Furthermore, interruption of CD28/B7 interaction with the soluble protein CTLA4lg caused inhibition of T cell proliferation (Linsley et al., 1992; Lenschow et al., 1992; Larsen et al., 1996). Further analogy to the effect of TIRC7 antibody targeting is provided by CTLA4lg selectively blocking Th1 and sparing Th2 lymphocyte responses (Mohammed et al., 1995).
  • TIRC7 does not share structural or sequence homology with any of the known T cell accessory molecules. Thus, TIRC7 may participitate in a distinct signaling pathway induced early in the course of T cell activation. This possibility is supported by recent reports that interference with pathways mediated by molecules other than the known costimulatory proteins can modulate the T cell response.
  • said peptide or polypeptide encoded by the above described polynucleotide comprises the amino acid sequence depicted in any one of SEQ ID NOS 3 to 9.
  • peptides comprising the above mentioned amino acid sequences correspond to parts of the extracellular domain of the TIRC7 protein and can advantageously be used for the generation of antibodies that are capable of inducing T-cell unresponsiveness.
  • the pharmaceutical composition of the invention comprises a soluble form of said peptide or polypeptide.
  • a soluble form of TIRC7 or a TIRC7 ligand is a truncated form of the molecule comprising an extracellular domain of the TIRC7 or a functional portion thereof.
  • a portion of the extracellular domain of TIRC7 which retains the ability to bind to a TIRC7 ligand can be used.
  • a portion of the extracellular domain of a TIRC7 ligand which retains the ability to bind to TIRC7 can be used.
  • fusion protein refers to a protein comprised of a first polypeptide from a first protein in contiguous amino acid sequence with a second polypeptide from a second protein. Fusion proteins can be made by standard recombinant DNA techniques wherein a nucleotide sequence encoding the first polypeptide is iigated in-frame to a nucleotide sequence encoding the second polypeptide, and these nucleotide sequences are expressed (e.g., using a recombinant expression vector introduced into a host cell) to produce the fusion protein.
  • a preferred fusion protein is an immunoglobulin fusion protein which includes an extracellular domain, or functional portion of TIRC7 or a TIRC7 ligand linked to an immunoglobulin heavy chain constant region (e.g., the hinge, CH2 and CH3 regions of a human immunoglobulin such as lgG1 ).
  • Immunoglobulin fusion proteins can be prepared, for example, according to the teachings of Capon, Nature 337 (1989), 525-531.
  • the antibody comprised in the pharmaceutical composition of the invention preferably has a specificity at least substantially identical to the binding specificity of the natural ligand of the TIRC7 protein of the invention, in particular if T-cell stimulation is desired.
  • Such an antibody can have a binding affinity of at least 10 5 M "1 , preferably not higher than 10 7 M "1 if T-cell stimulation is envisaged and advantageously up to 10 10 M "1 in case T-cell suppression should be mediated.
  • a T-cell suppressive antibody has an affinity of at least about 10 "7 M, preferably at least about 10 "9 M and most preferably at least about 10 '11 M;
  • a T-cell stimulating antibody has an affinity of less than about 10 '7 M, preferably less than about 10 "6 M and most preferably in order of 10 '5 M.
  • the binding site recognizing the tumor antigen has a high affinity in order to capture the target cells to be destroyed with high efficiency.
  • the binding affinity of the binding site recognizing the TIRC7 protein of the invention should be in the order of those of the natural TIRC7 ligand or of that usually found for the interaction of the T-cell costimulatory molecules with their receptor.
  • said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, humanized antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the production of chimeric antibodies is described, for example, in WO89/09622.
  • Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • the peptides, polypeptides and antibodies comprised in the pharmaceutical compositions of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds.
  • the linkage can be based on genetic fusion according to the methods known in the art and described above or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686.
  • the additional domain present in the fusion protein comprising the peptide, polypeptide or antibody employed in accordance with the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the peptide, polypeptide or antibody or vice versa.
  • the above described fusion protein may further comprise a cleavable linker or cleavage site for proteinases.
  • said further domain may be of a predefined specificity or function.
  • the literature contains a host of references to the concept of targeting bioactive substances such as drugs, toxins, and enzymes to specific points in the body to destroy or locate malignant cells or to induce a localized drug or enzymatic effect. It has been proposed to achieve this effect by conjugating the bioactive substance to monoclonal antibodies (see, e.g., N.Y. Oxford University Press; and Ghose, (1978) J. Natl. Cancer Inst. 61_:657-676).
  • polypeptides present in the pharmaceutical composition according to the invention may be further modified by conventional methods known in the art.
  • This allows for the construction of fusion proteins comprising the peptide, polypeptide or antibody of the invention and other functional amino acid sequences, e.g., nuclear localization signals, transactivating domains, DNA-binding domains, hormone-binding domains, protein tags (GST, GFP, h-myc peptide, FLAG, HA peptide) which may be derived from heterologous proteins.
  • administration of the composition of the invention can utilize unlabeled as well as labeled (poly)peptides or antibodies.
  • the peptides, polypeptides and antibodies can be administered labeled with a therapeutic agent.
  • agents can be coupled either directly or indirectly to the antibodies or (poly)peptides of the invention, see supra, and can be selected to enable drug release from the antigen at the target site.
  • therapeutic agents which can be coupled to the (poly)peptides and antibodies for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • the drugs which can be conjugated to the polypeptides of the invention include compounds which are classically referred to as drugs such as mitomycin C, daunorubicin, and vinblastine.
  • radioisotopically conjugated (poly)peptides or antibodies of the invention for, e.g., immunotherapy, certain isotopes may be more preferable than others depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters may be preferable to others. In general, and ⁇ particle-emitting radioisotopes are preferred in immunotherapy. Preferred are short range, high energy ⁇ emitters such as 12 Bi. Examples of radioisotopes which can be bound to the (poly)peptides and antibodies of the invention for therapeutic purposes are 125 l, 131 l, 90 Y, 67 Cu, 212 Bi, 212 At, 211 Pb, 47 Sc, 09 Pd and 188 Re.
  • Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. However, ricin is a toxic lectin which has been used immunotherapeutically. This is accomplished by binding the ⁇ -peptide chain of ricin, which is responsible for toxicity, to the polypeptide to enable site specific delivery of the toxic effect. Toxins are poisonous substances produced by plants, animals, or microorganisms that, in sufficient dose, are often lethal. Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This toxin consists of an ⁇ and ⁇ subunit which under proper conditions can be separated. The toxic A component can be bound to an antibody of the invention and be used for site specific delivery to the interacting T-cell.
  • the (poly)peptide and antibody of the invention can be used for the construction of fusion proteins of desired specificity and biological function.
  • the (poly)peptides and antibodies then optionally employed in accordance with the present invention of the invention as well as fusion protein thereof are expected to play an important therapeutic and scientific role in particular in the medical field, for example, in the development of new treatment approaches for T-cell related disorders such as certain forms of cancer and autoimmune diseases or as interesting tools for the analysis and modulation of the corresponding cellular signal transduction pathways.
  • said further domain comprises a molecule selected from the group consisting of effector molecules having a conformation suitable for biological activity, amino acid sequences capable of sequestering an ion, and amino acid sequences capable of selective binding to a solid support or to a preselected antigen.
  • Said domain may comprises an enzyme, toxin, receptor, binding site, biosynthetic antibody binding site, growth factor, cell-differentiation factor, lymphokine, cytokine, hormone, a remotely detectable moiety, anti-metabolite, a radioactive atom or an antigen.
  • Said antigen can be, e.g., tumor antigen, a viral antigen, a microbial antigen, an allergen, an auto-antigen, a virus, a microorganism, a polypeptide, a peptide or a plurality of tumor cells.
  • said sequence capable of sequestering an ion includes calmodulin, methallothionein, a fragment thereof, or an amino acid sequence rich in at least one of glutamic acid, aspartic acid, lysine, and arginine.
  • said polypeptide sequence capable of selective binding to a solid support can be a positively or negatively charged amino acid sequence, a cysteine-containing amino acid sequence, avidin, streptavidin, a fragment of Staphylococcus protein A, GST, a His-tag, a FLAG- tag, Lex A or c-myc as used in the appended examples.
  • Some of the effector molecules and amino acid sequences described above may be present in a proform which itself is either active or not and which may be removed, when, e.g., entering a certain cellular environment.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as T-cell costimulatory molecules or cytokines known in the art, or their inhibitors or activators depending on the intended use of the pharmaceutical composition.
  • the various polynucleotides and vectors encoding the above described peptides or polypeptides are administered either alone or in any combination using standard vectors and/or gene delivery systems, and optionally together with a pharmaceutically acceptable carrier or excipient.
  • the polynucleotide of the invention can be used alone or as part of a vector to express the (poly)peptide of the invention in cells, for, e.g., gene therapy or diagnostics of diseases related to T-cell disorders.
  • the polynucleotides or vectors of the invention are introduced into the cells which in turn produce the (poly)peptide.
  • polynucleotides or vectors may be stably integrated into the genome of the subject.
  • viral vectors may be used which are specific for certain cells or tissues and persist in said cells.
  • Suitable pharmaceutical carriers and excipients are well known in the art.
  • the pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of diseases, which are related to T-cell related immunodeficiencies and malignancies.
  • composition of the invention which comprises polynucleotide or vector of the invention in gene therapy.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808- 813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • the polynucleotides and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell populations.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.
  • suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno- associated viruses, among others.
  • nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991 ), 2726-2729). It is to be understood that the introduced polynucleotides and vectors express the gene product after introduction into said cell and preferably remain in this status during the lifetime of said cell.
  • cell lines which stably express the polynucleotide under the control of appropriate regulatory sequences may be engineered according to methods well known to those skilled in the art.
  • host cells can be transformed with the polynucleotide of the invention and a selectable marker, either on the same or separate plasmids.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • Such engineered cell lines are also particularly useful in screening methods for the detection of compounds involved in, e.g., T-cell activation or stimulation.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in tk “ , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC omithine decarboxylase
  • DFMO 2-(difluoromethyl)-DL-omithine
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising any one of the above described proteins, antibodies, (poly)peptides, polynucleotides, vectors or cells and optionally suitable means for detection.
  • the (poly)peptides and antibodies described above are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize said (poly)peptide are competitive and non-competitive immunoassays in either a direct or indirect format.
  • Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western blot assay.
  • the (poly)peptides and antibodies can be bound to many different carriers and used to isolate cells specifically bound to said polypeptides.
  • Examples of well-known earners include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • labels there are many different labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed hereinabove.
  • Said diagnostic compositions may also be used for methods for detecting expression of a polynucleotide of the invention by detecting the presence of mRNA coding for a TIRC7 membrane protein which comprises obtaining mRNA from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a polynucleotide of the invention under suitable hybridizing conditions (see also supra), detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the TIRC7 protein by the cell.
  • the invention comprises methods of detecting the presence of a TIRC7 membrane protein in a sample, for example, a cell sample, which comprises obtaining a cell sample from a subject, contacting said sample with one of the aforementioned antibodies under conditions permitting binding of the antibody to the TIRC7 protein, and detecting the presence of the antibody so bound, for example, using immuno assay techniques such as radioimmunoassay or enzymeimmunoassay.
  • one skilled in the art may specifically detect and distinguish polypeptides which are functional TIRC7 proteins from mutated forms which have lost or altered their T-cell stimulatory activity by using an antibody which either specifically recognizes a (poly)peptide which has TIRC7 activity but does not recognize an inactive form thereof or which specifically recognizes an inactive form but not the corresponding polypeptide having TIRC7 activity.
  • the antibodies of the present invention may also be used in affinity chromatography for purifying the TIRC7 membrane protein or above described (poly)peptides and isolating them from various sources.
  • the invention also encompasses a method for diagnosing in a subject a predisposition to a disorder associated with the expression of a TIRC7 allele which comprises isolating DNA from victims of the the disorder associated with the under- or over-expression of a TIRC7 protein; digesting the isolated DNA with at least one restriction enzyme; electrophoretically separating the resulting DNA fragments on a sizing gel; contacting the resulting gel with a nucleic acid probe as described above capable of specifically hybridizing to DNA encoding a TIRC7 protein and labeled with a detectable marker; detecting labeled bands on the gel which have hybridized to the labeled probe to create a band pattern specific to the DNA of victims of the disorder associated with the expression of a TIRC7 protein; preparing the subject's DNA according to the above-mentioned steps to produce detectable labeled bands on a gel; and comparing the band pattern specific to the DNA of victims of the disorder associated with the expression of a TIRC7 protein and the
  • the detectable markers of the present invention may be labeled with commonly employed radioactive labels, such as, for example, 32 P and 35 S, although other labels such as biotin or mercury as well as those described above may be employed as well.
  • radioactive labels such as, for example, 32 P and 35 S
  • Various methods well- known to the person skilled in the art may be used to label the detectable markers.
  • DNA sequences and RNA sequences may be labeled with 32 P or 35 S using the random primer method.
  • various methods well-known to the person skilled in the art may be employed for contacting the detectable marker with the sample of interest. For example, DNA-DNA, RNA-RNA and DNA-RNA hybridizations may be performed using standard procedures.
  • nucleic acids e.g., Southern and northern blotting, PCR, primer extension and the like.
  • the mRNA, cRNA, cDNA or genomic DNA obtained from the subject may be sequenced to identify mutations which may be characteristic fingerprints of TIRC7 mutations in disorders associated with the expression of TIRC7 or mutated versions thereof.
  • the present invention further comprises methods, wherein such a fingerprint may be generated by RFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNA may be amplified prior to analysis, the methods of which are well known in the art.
  • RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase T 1 ( RNase T 2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments on PAGE as described above or in the appended examples.
  • a suitable RNA-Enzyme for example RNase T 1 ( RNase T 2 or the like or a ribozyme
  • electrophoretically separating and detecting the RNA fragments on PAGE as described above or in the appended examples.
  • the pharmaceutical composition of the present invention comprises at least one second agent, preferably an agent which inhibits or activates T- cell stimulation depending on the intended use.
  • agents include, for example, molecules that are capable of blocking or mimicking receptor/ligand interaction or the like which leads to T-cell suppression and activation, respectively.
  • agents comprise those blocking the activity of, e.g., costimulatory molecules, integrins, Ig-superfamily molecules, selectins as well as drugs blocking chemokines and their respective receptor interactions, drugs blocking IL2/IL2-receptor interaction and other conventional immunosuppressive drugs such as IL-2R mAbs, IL-Toxins and IL-Muteins.
  • Examples for costimulatory molecules and their ligands are described in the prior art, e.g., in Schwartz, Cell 71 (1992), 1065-1068.
  • the interruption of the receptor/ligand interactions by using mAbs or soluble CTLA4lg for the interaction between CD28 to the B7-2 and CTLA4 to B7-1 and B7-2 are described in Blazar, J. Immunol. 157 (1996), 3250-3259; Bluestone, Immunity 2 (1995), 555-559; Linsley, Science 257 (1992), 792-95.
  • Examples for blocking the receptor/ligand interaction by using mAbs to CD40 or CD40L are reported by Burden, Nature 381 (1996), 434-435; Kirk, Proc. Natl. Acad. Sci.
  • CD2 antigen and its ligand LFA-3 are described in Bagogui Li et al., review in Adhesion Molecules, Fusion proteins, Novel Peptides, and Monoclonal Antibodies, Recent Developments in Transplantation Medicine, Vol. II, 1995, Physicians&Scientists Publishing Co., Inc. and blocking of their interaction by using of mAbs (anti-Leu-5b, OKT11 , T11 ) is reported in Brumberg, Transplantation 51 (1991) 219-225 or CD2.lgG1 fusion protein. The use of monoclonal Abs agains CD4 molecule is described in Cosimi, Surgery 108 (1990), 406-414.
  • Integrins and Ig- superfamiiy molecules include LFA-1 with its ligand ICAM-1, -2, -3, Mac-1 with ist ligand ICAM-1 , -3; ICAM-1 with its ligand LFA-1 , Mac-1 , CD43; ICAM-2 with ist ligand LFA-1 ; ICAM-3 with its ligand LFA-1 , Mac-1 ; VLA4 and VCAM-1 see, e.g., David, Adams, review in Adhesion Molecules, Fusion proteins, Novel Peptides, and Monoclonal Antibodies, Recent Developments in Transplantation Medicine, Vol.
  • VLA-4 mAbs to the alpha4 integrin chain can be used, betal integrin chain (CD29), or an activation - induced neo-epitope of VLA-4 as well as soluble VLA-4 ligands such as soluble fibronectin or its relevant peptide (GPEILDVPST), or soluble VCAM-1 or its relevant peptide.
  • More selectively blocking agents are antisense oligonucleotides, designed to selectively hybridize with cytoplasmic alpha4, betal , orVCAM-1 mRNA; Fedoseyeva, J. Immunol. 57 (1994), 606-612.
  • VCAM-1 drug pentoxifylline
  • Besler J. Leukoc. Biol. 40 (1986), 747-754.
  • VCAM-1 mAb, M/K-2, anti-murine for example could prolong allograft survival, Orosz, Transplantation, 56 (1993), 453-460.
  • CD62L L-selectin
  • CD62E E-selectin
  • CD62P P-selectin
  • Azathioprine, Mycophenylate, Mofetyl, Cyclosporin A, FK506, Corticosteroids may be used as decribed in Cosimi, Transplantation 32 (1981), 535-539; Shield,
  • any agent as defined above and referenced by way of example can be used in accordance with the pharmaceutical composition of the invention or the methods and uses described hereinbelow.
  • the polynucleotides, vectors, proteins, peptides, polypeptides, antibodies, cells, and pharmaceutical compositions of the invention can be used for methods and uses described for the above referenced T-cell costimulatory molecules, inhibitors and drugs.
  • the pharmaceutical composition of the invention is intended for use in organ transplantation, for the treatment of autoimmune, allergic or infectious diseases, or for the treatment of tumors.
  • An example for the use of the pharmaceutical composition of the invention for improving allograft or xenograft tolerance is described with respect to administration of an LFA-3 and CD2 binding protein, respectively, in WO93/06852.
  • this invention pertains to methods for modulating (antigen-specific) T- cell unresponsiveness.
  • T-cell unresponsiveness refers to a reduction in or lack of T-cell proliferation, lymphokine secretion or induction of effector functions by a T-cell upon exposure to the antigen (or antigenic portion).
  • the pharmaceutical compositions of this invention provide a means for inducing, maintaining or reversing unresponsiveness of a T-cell to an antigen in vitro or in vivo. Accordingly, the compounds of this invention are particularly useful for modulating (antigen-specific) T-cell unresponsiveness.
  • modulation is intended to include both inducing and maintenance of an unresponsive state and reversal of an unresponsive state, i.e., restoration of T-cell responsiveness.
  • the present invention relates to an in vitro method for inducing or maintaining unresponsiveness of a T-cell to an antigen comprising contacting the T-cell with an agent which inhibits stimulation of the T-cell through a TIRC7 membrane protein.
  • Recipes for how to carry out methods for modulation T-cell unresponsiveness are generally known to the skilled person and are described in, e.g., WO95/24217 and references cited therein.
  • the present invention also relates to the use of an agent which inhibits T-cell stimulation through a TIRC7 membrane protein for the preparation of a pharmaceutical composition for inducing or maintaining T-cell unresponsiveness to an antigen in a subject.
  • an agent which inhibits T-cell stimulation through a TIRC7 membrane protein for the preparation of a pharmaceutical composition for inducing or maintaining T-cell unresponsiveness to an antigen in a subject.
  • the methods and uses of the invention may be used with primed or unprimed T-cells depending on what is intended by the person skilled in the art.
  • the agent blocks an interaction of the TIRC7 membrane protein with its ligand.
  • the results of the experiments performed within the scope of the present invention suggest the existence of a ligand interacting with the TIRC7 protein and thereby stimulating T- cell proliferation. Blocking said interaction, e.g. with antibodies or soluble (poly)peptides derived from the TIRC7 membrane protein should result in T cell unresponsiveness.
  • the agent is a polynucleotide, a vector, a cell, peptide or polypeptide, or antibody described hereinbefore.
  • the above described method or use further comprise the use of a second agent as defined above.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a first agent which stimulates a T-cell through a TIRC7 membrane protein, and optionally a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a first agent which stimulates a T-cell through a TIRC7 membrane protein, and optionally a pharmaceutically acceptable carrier.
  • the agent that stimulates the T-cell through the TIRC7 membrane protein is expected to markedly enhance the proliferation of (activated) T-cells and thus is capable of augmenting the immune response. Examples for this type of "Vaccine" is described, e.g., in WO91/11194 and in the literature, e.g., referred to above.
  • Such agents also comprise promoters which can be inserted in front of the coding region of the TIRC7 protein encoding gene, e.g., via gene transfer and homologous recombination in the 5' untranslated region of the gene, see also supra.
  • promoter may be regulated and thus permit the controlled expression of the TIRC7 protein in certain cells.
  • said agent is a ligand of the TIRC7 membrane protein or is at least one anti-TIRC7 membrane protein antibody described above.
  • the pharmaceutical composition further comprises a second agent which stimulates T-cell proliferation, for example IL-2, IL-4 or an agent which stimulates a T-cell through a CD2, CD28, CD40 or CTLA4 surface receptor.
  • a second agent which stimulates T-cell proliferation for example IL-2, IL-4 or an agent which stimulates a T-cell through a CD2, CD28, CD40 or CTLA4 surface receptor.
  • the present invention relates to an in vitro method for restoring responsiveness to an antigen by a T-cell which is unresponsive to the antigen, comprising contacting the T-cell in the presence of the antigen with a first agent which stimulates the T-cell through a TIRC7 membrane protein.
  • said TIRC7 ligand may be preferably expressed on the cell surface by introducing into the cell a nucleic acid molecule encoding the TIRC7 ligand in a suitable form for expression of the TIRC7 ligand on the cell surface.
  • the cell is a tumor cell.
  • Vectors and methods for the introduction of such, nucleic acid molecules are well known to the skilled person and are also described, e.g., above.
  • the method of the invention can further comprise contacting the T-cell with a second agent as defined for the pharmaceutical compositions above.
  • the T-cell is contacted with the second agent prior to being contacted with the first agent.
  • the second agent preferably CD2, CD28, CTLA4 ligand or CD40 is expressed on the cell surface by introducing into the cell a nucleic acid molecule encoding the CD2, CD28, CTLA4 ligand or CD40 in a form suitable for expression of said ligand on the cell surface.
  • the present invention relates to the use of a first agent as defined above, which is capable of stimulating a T-cell through a TIRC7 membrane protein for the preparation of a pharmaceutical composition for stimulating a T-cell response to a tumor cell in a subject with a tumor.
  • a first agent as defined above, which is capable of stimulating a T-cell through a TIRC7 membrane protein for the preparation of a pharmaceutical composition for stimulating a T-cell response to a tumor cell in a subject with a tumor.
  • agents comprise, for example, also bispecific antibodies described supra.
  • the tumor cell is modified to express a TIRC7 ligand and/or a CD2, CD28, CTLA4 ligand or CD40 preferably on the tumor cell surface.
  • the tumor cell is obtained from the subject, modified ex vivo to form a modified tumor cell and said modified tumor cell is used for the preparation of a pharmaceutical composition which is designed for administration to the subject.
  • the T-cells are obtained from a subject, contacted with IL-2 or IL-4 ex vivo and said modified T-ceils are used for the preparation of pharmaceutical composition which is designed for the administration to the subject.
  • the uses, methods and pharmaceutical compositions are intended to be applied to a subject who is a recipient of bone marrow transplant or peripheral stem cell transplant.
  • the pharmaceutical composition is designed for contacting with bone marrow or peripheral stem cell prior to transplantation into the recipient.
  • the methods and uses of the present invention are applied in organ graft transplantation, peripheral stem cell transplantation or for the treatment of auto-immune diseases or allergy.
  • the invention relates to a method for identifying T-cell activating or co- stimulating compounds or for identifying inhibitors of T-cell activation and stimulation comprising
  • compound in the method of the invention includes a single substance or a plurality of substances which may or may not be identical.
  • Said compound(s) may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compounds may be known in the art but hitherto not known to be capable of inhibiting T-cell activation or not known to be useful as a T-cell costimulatory factor, respectively.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into the cell.
  • a sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. It can then be determined whether said sample or compound displays the desired properties by methods known in the art such as described herein and in the appended examples. Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s).
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art (see, e.g., EP-A-0 403 506) or by using and modifying the methods as described in the appended examples.
  • the person skilled in the art will readily recognize which further compounds and/or cells may be used in order to perform the methods of the invention, for example, B-cells, interieukins, or enzymes, if necessary, that, e.g., convert a certain compound into the precursor which in turn stimulates or suppresses T-cell activation or that provide for (co)stimulatory signals.
  • Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
  • Compounds which can be used in accordance with the method of the present invention include peptides, proteins, nucleic acids including cDNA expression libraries, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds can also be functional derivatives or analogues of known T-cell activators or inhibitors. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
  • said derivatives and analogues can be tested for their effects according to methods known in the art or as described, for example, in the appended examples.
  • peptidomimetics and/or computer aided design of appropriate activators or inhibitors of T-cell activation can be used, for example, according to the methods described below.
  • Appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the TIRC7 protein by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.
  • peptidomimetic combinatorial libraries can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein and in the appended examples. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.
  • the present invention provides methods for identifying compounds which are capable of modulating T-cell mediated immune responses. Accordingly compounds identified in accordance with the method of the present invention to be inhibitors and activators, respectively, of T-cell stimulation or activation are also within the scope of the present invention.
  • Compounds found to activate T-cell mediated responses may be used in the treatment of cancer and related diseases. In addition, it may also be possible to specifically inhibit viral diseases, thereby preventing viral infection or viral spread.
  • Compounds identified as suppressors of T-cell activation or stimulation can be used, e.g., for treating skin conditions (see, e.g., WO93/06866) or in allogenic or xenogenic cell or organ transplantation in order to avoid graft rejection; see also supra.
  • the compounds identified or obtained according to the method of the present invention are thus expected to be very useful in diagnostic and in particular for therapeutic applications.
  • the invention relates to a method for the production of a pharmaceutical composition
  • a method for the production of a pharmaceutical composition comprising formulating and optionally synthesizing the compound identified in step (b) of the above described method of the invention in a pharmaceutically acceptable form.
  • the present invention generally relates to a method of making a therapeutic agent comprising synthesizing the proteins, (poly)peptides, polynucleotides, vectors, antibodies or compounds according to the invention in an amount sufficient to provide said agent in a therapeutically effective amount to the patient. Methods for synthesizing these agents are well known in the art and are described, e.g. above.
  • the therapeutically useful compounds identified according to the method of the invention may be administered to a patient by any appropriate method for the particular compound, e.g., orally, intravenously, parenterally, transdermally, transmucosally, or by surgery or implantation (e.g., with the compound being in the form of a solid or semi-solid biologically compatible and resorbable matrix) at or near the site where the effect of the compound is desired.
  • Therapeutic doses are determined to be appropriate by one skilled in the art, see also supra.
  • Such useful compounds can be for example transacting factors which bind to the TIRC7 protein of the invention. Identification of transacting factors can be carried out using standard methods in the art (see, e.g., Sambrook, supra, and Ausubel, supra). To determine whether a protein binds to the TIRC7 protein of the invention, standard native gel-shift analyses can be carried out. In order to identify a transacting factor which binds to the TIRC7 of the invention, the polypeptides and peptides of the invention can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
  • the transacting factor modulation of its binding to the TIRC7 protein of the invention can be pursued, beginning with, for example, screening for inhibitors against the binding of the transacting factor to the TIRC7 protein of the present invention.
  • Activation or repression of TIRC7 specific genes could then be achieved in subjects by applying the transacting factor (or its inhibitor) or the gene encoding it, e.g., in a vector described in the embodiments hereinbefore.
  • the active form of the transacting factor is a dimer, dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
  • further components in the pathway leading to activation e.g.
  • TIRC7 protein of the present invention can then be identified. Modulation of the activities of these components can then be pursued, in order to develop additional drugs and methods for modulating the expression or activity of the TIRC7 protein of the present invention.
  • the described polynucleotides may also be used for several other applications, for example, for the identification of nucleic acid molecules which encode proteins which interact with the TIRC7 protein described above. This can be achieved by assays well known in the art, for example, as described in Scofield (Science 274 (1996), 2063- 2065) by use of the so-called yeast "two-hybrid system". In this system the (poly)peptide encoded by the polynucleotides according to the invention or a smaller part thereof is linked to the DNA-binding domain of the GAL4 transcription factor.
  • a yeast strain expressing this fusion protein and comprising a lacZ reporter gene driven by an appropriate promoter, which is recognized by the GAL4 transcription factor, is transformed with a library of cDNAs which will express animal, preferably mammal proteins or peptides thereof fused to an activation domain.
  • a peptide encoded by one of the cDNAs is able to interact with the fusion protein comprising a (poly)peptide of the invention, the complex is able to direct expression of the reporter gene.
  • the polynucleotide according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with TIRC7 proteins.
  • the present invention relates to the use of the polynucleotide, the vectors, peptides, polypeptides, antibodies and cells of the invention as well as compounds identified in accordance with a method of the invention described hereinabove for the preparation of a pharmaceutical composition for the treatment of diseases involving T-cell activation and associated with Th1 and Th2 immune response, for the treatment of acute and chronic rejection of allo-and xeno organ transplants and bone marrow transplantation, for the treatment of rheumatoid arthritis, lupus erythramatodes, multiple sklerosis, encephalitis, vasculitis, diabetes mellitus, pancreatitis, gastritis, thyroiditis, for the treatment of maligne disorders of T, B or NK cells, for the treatment of asthma, lepramatosis, Helicobacter pylori associated gastritis or for the treatment of skin tumors, adrenal tumors or lung tumors.
  • polynucleotides, vectors, cells, proteins, (poly)peptides, antibodies, inhibitors, activators, pharmaceutical compositions, uses and methods of the invention can be used for the treatment of all kinds of diseases hitherto unknown as being related to or dependent on the modulation of T-cells.
  • the pharmaceutical compositions, methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.
  • FIG. 1 Identification of TIRC7 from alloactivated T cells.
  • A Differential display identification of a 350 bp transcript upregulated 24 hours after alloactivation of human T lymphocytes. Each lane shows a mRNA expression pattern from a one-way MLR at either 0 or 24 h after activation. Two different MLRs (a and b) exhibited similar patterns of gene expression.
  • B Nucleotide sequence of TIRC7 cDNA. The cDNA and deduced 614 amino acid sequences of the TIRC7 transcript is shown. Predicted transmembrane regions are underlined and bold.
  • Figure 2 Predicted secondary structure of TIRC7.
  • TIRC7 protein contains seven transmembrane spanning domains (TM).
  • Peptides (P1 - P7) synthesized according to sequences in the putative intracellular amino terminus (NH3), extracellular carboxy terminus (COOH), and the largest intracellular (IC) and extracellular (EC) loop were used to raise rabbit anti-TIRC7 polyclonal antibodies.
  • Anti-TIRC7 antibodies with T cell response modulatory effect are given in the box.
  • TIRC7 is upregulated in T cell activation and is localized on the cell membrane.
  • TIRC7 mRNA expression is upregulated in allostimulated human T cells.
  • MLR-0 and MLR-24 indicate time points 0 h and 24 h, respectively, after coincubation of allogenic responder and stimulator lymphocytes.
  • Figure 4 Localization of TIRC7 protein.
  • a single 75 kDa protein is detected by anti-TIRC7 antibodies (Ab79 is shown) predominantly in membrane protein extracts of human lymphocytes. Binding of Ab79 to TIRC7 is abolished in the presence of the respective peptide P2 (Ab+P2).
  • A Anti-TIRC7 antibodies (Ab73, Ab76, Ab79) directed against extracellularly located TIRC7 peptides (P4, P6, P2) inhibit proliferation in alloantigen stimulated T cells as determined by [ ⁇ Hj-thymidine incorporation. Inhibition was diminished when antibodies were preincubated with their respective peptides. Proliferation in a MLR after 24 h is displayed as positive control. Proliferation was not affected by preimmune serum (control Ab). Each bar represents mean and SD from seven independent experiments.
  • B Inhibition of proliferation by exogenous TIRC7 protein. In a one-way MLR in vitro translated TIRC7 protein inhibited proliferation in a dose-dependent manner. Protein dilutions are indicated.
  • FIG. 7 Histological analysis of kidney allografts at day 7 post-transplantation.
  • A Kidney allografts of rats receiving control antibodies showed severe tissue destruction and diffuse mononuclear infiltration which was similar to histological findings in the kidney allografts of untreated animals.
  • Example 1 Cloning of a novel membrane protein (TIRC7) encoding a gene that is differentially expressed in alloactivated human T cells
  • PBLs peripheral blood lymphocytes
  • Responder PBLs were stimulated with equal numbers of irradiated (3000 rad, 13 min) stimulator PBLs.
  • Cells were co-cultured for 24 h in tissue flasks at an initial concentration of 10 6 cells/ml for RNA isolation.
  • the primers for the PCR amplification were: 5'- GACGGAACAGCTTC -3" (SEQ ID No. 10) and 5'- TGCGTCTGGTTCT-3' (SEQ ID No. 11).
  • the PCR products were stored at 4°C and separated by electrophoresis in 6% polyacrylamide-urea gels, transferred to filter paper, dried, and autoradiographed.
  • the differentially expressed cDNA fragment was excised from the gel, eluted, reamplifed, cloned into pBluescriptSK * plasmid, and sequenced at the Howard Hughes Biopolymers Research Facility or the Dana Farber Cancer Institute Biopolymer Facility at Harvard Medical School. Homology searches were performed using BLAST at NCBI.
  • plaque lifts of 1,200,000 independent cDNA clones were hybridized with a 32 P-labeled cDNA for 24 hrs at 42°C in 40% formamide, 10% dextran sulfate, 4 X SSC (1 X SSC consists of 150 mM NaCI, 15 mM sodium citrate, pH 7.0), 0.8 X Denhardt's solution (1 X Denhardt's contains of 0.02% polyvinylpyrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), and 20 mg salmon sperm DNA.
  • 4 X SSC (1 X SSC consists of 150 mM NaCI, 15 mM sodium citrate, pH 7.0), 0.8 X Denhardt's solution (1 X Denhardt's contains of 0.02% polyvinylpyrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.5% sodium dodecyl sulfate (S
  • the filters were washed twice for 20 min at room temperature with 2 X SSC, 10% SDS and for 30 min at 65°C with 0.2 X SSC, 10% SDS followed by autoradiography. Three positive clones were selected and plaque purified. cDNA was sequenced in both directions using a primer walking strategy. A PAC genomic library was screened using a 2 kb cDNA probe containing the ORF cDNA of TIRC7, and the entire genomic cDNA of TIRC7 and OC116 was bidirectinally sequenced.
  • TIRC7 T cell immune response cDNA7; Gen Bank Accession Number: AF025374
  • Figure 1B a second cDNA of TIRC7 was cloned (SEQ ID No. 12) encoding an amino acid sequence that is identical to that of the protein encoded by the other TIRC7 cDNA except for one amino acid substitution at amino acid position 121 (Arg ⁇ Gin). It was therefore concluded that both cDNAs represent alleles of the TIRC7 encoding gene.
  • TIRC7 Hydrophobicity analysis of the protein sequence revealed seven hydrophobic domains, compatible with transmembrane spanning domains.
  • the N-terminus of TIRC7 lacks a consensus signal peptide sequence and followed by seven hydrophobic domains predicting a topology of an intracellular N-terminus and extracellularly oriented C-domain ( Figure 2).
  • TIRC7 contains multiple putative sites of post-translational modification including phosphorylation sites for PKC (at amino acids 58, 98, and 148) and PKA (at amino acid 21 ), as well as N-linked glycosylation sites (at amino acid 267 and 287). No amino acid homology was found with any proteins known to be involved in T cell activation.
  • VPP vacuolar proton pump H + -ATPase
  • TIRC7 a recently reported human osteoclast specific cDNA, named OC116 (Li et al., 1996), are alternatively spliced transcripts of the same gene.
  • the function of OC116 is so far unknown and the 2640 nt mRNA, encoding a 814 residue protein, was demonstrated to be exclusively expressed in human osteoclast cells.
  • the regions of strongest homology between TIRC7 and these putative VPPs are predominantly in their predicted transmembrane domains and the C-termini.
  • TIRC7 belongs to a larger family of structurally related membrane proteins whose functions have not been clearly elucidated.
  • Northern blot analysis of total RNA from alloantigen activated lymphocytes was performed.
  • Northern blots were prepared with 7-10 ⁇ g of total RNA as described previously (Kojima et al., 1996).
  • Poly(A) + Northern blots containing RNA from various human tissues were purchased from Clontech.
  • Northern blots were probed with the full-length TIRC7 cDNA or a TIRC7- specific cDNA fragment (nt 52-391).
  • TIRC7 expression was transient with no increase at 1 h, peak expression at 24 h, and a return to baseline at 72 h.
  • an additional MLR was performed using stimulators depleted of T cells.
  • PBLs were exposed to concanavalin A (10 ng/ml), phytohemagglutinin (PHA) (20 mg/ml), Staphylococcus aureus enterotoxin B (10 ⁇ g/ml), OKT3-mAb (10 mg/ml), cyclosporine A (1 mg/ml) or rlL-2 (10 U/ml).
  • PHA phytohemagglutinin
  • PHA phytohemagglutinin
  • Staphylococcus aureus enterotoxin B (10 ⁇ g/ml
  • OKT3-mAb 10 mg/ml
  • cyclosporine A (1 mg/ml) or rlL-2 (10 U/ml
  • RNA isolation from CD4 + and CD8 * human T cells PBLs were incubated with immunomagnetic beads coated with anti-CD4 or anti-CD8 IgG and then subjected to magnetic separation.
  • TIRC7 mRNA expression was found to be increased in responder T cells 24 h after co-culture.
  • Cyclosporine A Cyclosporine A (Cyc A), an inhibitor of the calcineurin dependent T cell activation pathways, blocked the induction of TIRC7 in a MLR ( Figure 3B).
  • exogenous IL-2 was a potent inducer of TIRC7 expression, whereas a modest increase in TIRC7 expression was observed with Staphylococcus aureus enterotoxin B (SEB) or OKT3-mAb stimulation after 24 h, though OKT3-mAb increased TIRC7 expression after 48 h to similar levels induced by alloantigen.
  • SEB Staphylococcus aureus enterotoxin B
  • OKT3-mAb increased TIRC7 expression after 48 h to similar levels induced by alloantigen.
  • ConA concanavalin A
  • PHA phytohemagglutinin
  • TIRC7 is almost exclusively expressed in immune tissues and exhibits high levels of mRNA expression in spleen, lymph nodes, peripheral blood, and appendix, whereas lower levels of expression are observed in bone marrow, fetal liver and thymus, respectively.
  • TIRC7 was also detected in CD4 + and CD8 + lymphocytes but not in EBV-transformed primary B cells, Burkitt's lymphoma cells, EBV-infected Burkitt's lymphoma cells, and resting or activated Jurkat cells.
  • the membranes were incubated with primary (see Example 2, infra) and secondary antibody for 1 h each, and bound primary antibody was detected by horseradish peroxidase-conjugated secondary antibody followed by enhanced chemilumi- nescence (Boehringer).
  • Polyclonal Ab79 (see Example 2, infra) was used at 1 :5000 dilution.
  • the secondary antibody (anti-rabbit-lgG) conjugated with peroxidase was used at 1 :2000 dilution.
  • TIRC7 membrane preparations from CHO cells and COS-7 cells ( Figure 4B) stably and transiently transfected with a c- myc tagged TIRC7 expression vector, respectively.
  • the full-length TIRC7 ORF was cloned upstream of a c-myc epitope sequence to create a fusion protein construct in a mammalian expression vector (Progmega).
  • Transient transfection of COS7 cells and stable transfection of CHO cells was performed by lipofectamine transfection method as described (Sch ⁇ lein et al., 1996).
  • An anti-c-myc antibody InVitrogen was used to detect the protein.
  • TIRC7 localization to the cell membrane was confirmed by confocal microscopy and flow cytometric analysis in human lymphocytes as well as in stably transfected CHO cells.
  • Example 2 TIRC7 mediates an essential signal during early events of T cell activation
  • P1-P7 Purified synthetic peptides (Laboratories of Henklein, Berlin, Germany) were used for immunization of rabbits (Seramun, Berlin, Germany). Animals were boostered after three and six weeks. A total of 14 polyclonal antibodies were prepared against 7 different peptides (P1-P7). The pooled antisera were purified by affinity chromatography after binding of peptide to BSA. All antibodies were tested by ELISA with their respective peptides. As shown in Figure 5A, three of the antibodies Ab73, Ab76 and Ab79, which were directed against the extracellularly located domains P4, P6, and P2 (SEQ ID Nos.
  • responder PBLs were plated in the presence of an equal number of irradiated-stimulator cells (total of 2 x 10 6 cells / ml) with either media alone, antibodies or control serum into each well of a round-bottomed 96-well microtiter plate in a final culture volume of 200 ⁇ l.
  • Anti-TIRC7 anti-sera were added in 1 :500 dilutions to MLR. The plates were incubated at 37°C, 5% CO 2 and pulsed for the final 18 h of the culture with 1 ⁇ Ci [ 3 H]-thymidine (ICN Biochemicals) per well.
  • the cytokine expression for IL-2, IL-4 and interferon ⁇ y were detected in culture supernatants by ELISA.
  • Commercial kits were used for IL-2 (Laboserv), IL-4 (Laboserv) and IFN- ⁇ (Medgenix).
  • the anti-TIRC7 antibodies inhibited T cell proliferation in a dose dependent manner. Inhibition was diminished when the respective peptides were added to the reaction to specifically neutralize the antibody. The antibodies had no effect when added 24 h - 72 h after initiating the MLR, indicating that the TIRC7 mediated signal was specific for an early event in the T cell response.
  • These three anti-TIRC7 antibodies also caused efficient inhibition of T-cell activation induced by ConA, PHA, and OKT3-mAb, respectively.
  • TIRC7 Membrane proteins associated with T cell activation are often involved in ligand- receptor interactions that can be blocked by exogenous soluble protein, as has been demonstrated by blocking of CD28/B7 interaction with the soluble protein CTLA4lg (Linsley et al., 1992).
  • In vitro translated TIRC7 protein was therefore tested for its ability to inhibit the MLR by adding it to MLR cultures at time 0 at dilutions of 1 :200, 1:300, 1:600. 2 ⁇ g of TIRC7 cDNA were translated in an in vitro translation TNT lysate system (Promega) containing ⁇ S methionine (ICN). The product was visualized by SDS-page (11%) and autoradiography.
  • TIRC7 protein was synthesized in vitro in the presence of microsomal membranes. The in vitro translation mixture was then suspended in 500 ⁇ l PBS and dialyzed against PBS for 24 h. As shown in Figure 5B, exogenous TIRC7 protein significantly suppressed the proliferation of alloreactive T cells in a dose-dependent manner. In control experiments, no inhibition was seen using either vector alone ( Figure 5B) or another unrelated cDNA in the in vitro translation reaction mixture.
  • Example 3 Targeting of the TIRC7 mediated signal inhibits Th1 specific cytokine expression, which is reversed by exogenous rlL-2
  • TIRC7 To further differentiate whether signals mediated by TIRC7 differentially affect T cell subsets, human T cells were challenged with either OKT3-mAb, ConA or PHA, and the cytokine profiles specific for Th1 and Th2 lymphocyte subsets were analyzed in the presence and absence of anti-TIRC7 antibodies. As shown in Figure 5C, a significant decrease of the Th1-specific cytokines IL-2 and IFN- ⁇ was observed at 48 h in all cultures of PHA stimulated lymphocytes.
  • Example 4 TIRC7 antibody targeting significantly prolongs renal allograft survival in vivo
  • the effect of modulating the TIRC7 mediated signal was studied in an animal model featuring kidney transplantation from Wistar Furth to Lewis rats.
  • Male inbred rats 200-250 g (Harlan Winkelmann, Germany) were used throughout the experiment.
  • Wistar Furth rats (WF, RT1 U ) were grafted into bilaterally nephrectomized Lewis rats (LEW, RT1 1 ) using microsurgical techniques; ischemic time was 30 ⁇ 5 min.
  • Cryostat sections were fixed in formalin. The fixed tissue was paraffin embedded, and tissue sections were stained with hematoxylin and eosin.
  • anti-human TIRC7 antibodies were tested for their ability to inhibit the proliferation of Lewis rat lymphocytes stimulated with irradiated Wistar Furth rat lymphocytes in vitro. Ab73 was shown to profoundly block rat T cell proliferation.
  • Anti-TIRC7 antibody significantly prolonged the graft survival time of treated animals (p ⁇ 0, 001 ).
  • TIRC7 is a novel T-cell accessory protein
  • TIRC7 represents a novel protein that plays an essential role in T cell activation. Early after stimulation of the T cell receptor the level of TIRC7 mRNA is transiently increased. This increase in expression appears to rely on an IL-2 dependent pathway, as upregulation of TIRC7 mRNA is also observed after incubation of T cells with IL-2 and blockade of TIRC7 upregulation is achieved with cyclosporine A. T cell stimulation by mitogens such as ConA or PHA consistently fail to upregulate TIRC7 expression. The pattern of tissue expression suggests that TIRC7 is a product of mature lymphoid cells, as TIRC7 is expressed in all lymphoid tissues with low expression only in thymus, bone marrow and fetal liver.
  • the TIRC7 protein is predominantly expressed on the cell membrane, consistent with a target for an external ligand.
  • the seven transmembrane domain structure predicts three extracellular loops and an extracellularly oriented carboxy terminus.
  • Anti-TIRC7 antibodies directed against the extracellular domains, but not those recognizing predicted intracellular domains of the protein, are able to efficiently suppress the proliferation of T cells in response to alloactivation in a mixed lymphocyte culture or in response to mitogens.
  • the inhibitory effect of anti-TIRC7 antibodies on T cells induced by a variety of different stimulatory pathways suggests that TIRC7 plays a central role in T cell activation.
  • TIRC7 shares 38% amino acid homology with J6B7, a protein isolated from a mouse T cell line (Lee et al., 1990). Like TIRC7, J6B7 exhibits considerable homology to the putative rat H + -ATPase subunit VPP116 (Manolson et al., 1992). In vitro translated J6B7 protein was demonstrated to inhibit mouse T cell proliferation in a MLR by 89%, which is comparable with the results obtained with in vitro translated soluble TIRC7 protein in human MLR in the present study.
  • Antibody targeting of TIRC7 has a selective inhibitory effect on the Th1 lymphocyte subset, as evidenced by the inhibition of IL-2 and IFN- ⁇ , but not IL-4, cytokine production.
  • anti-TIRC7 antibody treatment the cells appear to remain in an unresponsive, but functional, state since exogenous recombinant IL-2 reversed the antiproliferative effect of the anti-TIRC7 antibodies.
  • the ability of an anti-TIRC7 antibody to prevent allograft rejection in the in vivo model of rat kidney transplantation reflects the findings obtained in the in vitro studies.
  • the effects of antibody targeting of TIRC7 are quite similar to those observed by targeting of costimulatory molecules.
  • TIRC7 does not share structural or sequence homology with any of the known T cell accessory molecules. Thus, TIRC7 may participitate in a distinct signaling pathway induced early in the course of T cell activation. Given the functional similarities between TIRC7 and the known T cell accessory molecules, it is expected that the structural novelty of TIRC7 will contribute to the understanding of distinct mechanisms in the T cell response. Moreover, the striking capacity of anti-TIRC7 antibody to significantly prolong allograft survival in vivo provide a novel approach for a selective inhibition of undesired T cell activation in human organ transplantation and autoimmune diseases.
PCT/EP1998/005462 1997-08-29 1998-08-28 T-cell membrane protein (tirc7), peptides and antibodies derived therefrom and uses thereof WO1999011782A1 (en)

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DE69820449T DE69820449T2 (de) 1997-08-29 1998-08-28 T-zell-membranprotein (tirc7), davon abgeleitete peptide und antikörper und ihre verwendungen
AU92654/98A AU751151C (en) 1997-08-29 1998-08-28 T-cell membrane protein (TIRC7), peptides and antibodies derived therefrom and uses thereof
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AT98945293T ATE256185T1 (de) 1997-08-29 1998-08-28 T-zell-membranprotein (tirc7), davon abgeleitete peptide und antikörper und ihre verwendungen
US11/126,866 US7863418B2 (en) 1997-08-29 2005-05-10 T-cell membrane protein (TIRC7) and peptides derived therefrom
US11/126,841 US20050271659A1 (en) 1997-08-29 2005-05-10 Novel T-cell membrane protein (TIRC7), peptides and antibodies derived therefrom and uses thereof
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EA007814B1 (ru) * 2000-03-01 2007-02-27 Чей 13 Медикал Рисерч Груп Н.В. Пептиды, полученные из казеина, и их использование в терапии
WO2001082948A2 (en) * 2000-05-03 2001-11-08 Genpat77 Pharmacogenetics Ag Uses of tgap7 for the modulation of leucocyte activation
WO2001082948A3 (en) * 2000-05-03 2002-05-23 Genpat77 Pharmacogenetics Ag Uses of tgap7 for the modulation of leucocyte activation
WO2002036149A2 (en) * 2000-10-30 2002-05-10 Nalan Utku Methods for obtaining inhibitors of t-cell membrane protein (tirc7) ligand binding and uses thereof
AU2002221775B2 (en) * 2000-10-30 2006-11-23 Genpat77 Pharmacogenetics Ag Methods for obtaining inhibitors of t-cell membrane protein (TIRC7) ligand binding and uses thereof
WO2002036149A3 (en) * 2000-10-30 2003-08-28 Nalan Utku Methods for obtaining inhibitors of t-cell membrane protein (tirc7) ligand binding and uses thereof
US8110364B2 (en) 2001-06-08 2012-02-07 Xdx, Inc. Methods and compositions for diagnosing or monitoring autoimmune and chronic inflammatory diseases
WO2003025000A2 (en) * 2001-09-17 2003-03-27 Nalan Utku Peptides capable of modulating immune response
WO2003025000A3 (en) * 2001-09-17 2003-08-07 Nalan Utku Peptides capable of modulating immune response
WO2003045420A2 (en) * 2001-11-27 2003-06-05 Nalan Utku Use of t-cell immune response cdna 7 (tirc7) for modulation of angiogenesis and/or neovascularization
WO2003045420A3 (en) * 2001-11-27 2004-05-13 Nalan Utku Use of t-cell immune response cdna 7 (tirc7) for modulation of angiogenesis and/or neovascularization
WO2003054018A3 (en) * 2001-12-21 2004-03-04 Genpat77 Pharmacogenetics Ag Anti-tirc7 antibodies in therapy of inflammatory diseases
WO2003054018A2 (en) * 2001-12-21 2003-07-03 Genpat77 Pharmacogenetics Ag Anti-tirc7 antibodies in therapy of inflammatory diseases
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US9399679B2 (en) 2001-12-21 2016-07-26 Nalan Utku Therapeutic anti-TIRC7 antibodies for use in immune related and other diseases
WO2003054019A2 (en) * 2001-12-21 2003-07-03 Genpat77 Pharmacogenetics Ag Therapeutic monoclonal anti-tirc7 antibodies for use in immune related and other diseases
US7794715B2 (en) 2001-12-21 2010-09-14 Cellact Pharma Gmbh Anti-TIRC7 antibodies in therapy of inflammatory diseases
WO2003066091A2 (en) * 2002-02-04 2003-08-14 Nalan Utku Compositions and methods for modulation of effects on phagocyte and lymphoid cell populations employing tirc7
WO2003066091A3 (en) * 2002-02-04 2004-02-05 Nalan Utku Compositions and methods for modulation of effects on phagocyte and lymphoid cell populations employing tirc7
WO2003091285A1 (en) * 2002-04-26 2003-11-06 Genpat77 Pharmacogenetics Ag Bispecific molecules for use in therapy and diagnosis of immune related and other diseases
WO2003093318A1 (en) * 2002-04-29 2003-11-13 Genpat77 Pharmacogenetics Ag Novel antibody binding tcr and tirc7 and its use in therapy and diagnosis
WO2006122791A2 (en) * 2005-05-17 2006-11-23 Genpat77 Pharmacogenetics Ag Tirc7 as marker for detecting early immune activation
WO2006122791A3 (en) * 2005-05-17 2007-02-15 Genpat77 Pharmacogenetics Ag Tirc7 as marker for detecting early immune activation
US8642512B2 (en) * 2005-05-17 2014-02-04 Cellact Pharma Gmbh Method of detecting early immune activation
US20140154279A1 (en) * 2005-05-17 2014-06-05 Cellact Pharma Gmbh Peptides capable of modulating the function of tirc7
US9096654B2 (en) 2005-05-17 2015-08-04 Cellact Pharma Gmbh Peptides capable of modulating the function of TIRC7
WO2006122792A1 (en) * 2005-05-17 2006-11-23 Genpat77 Pharmacogenetics Ag Peptides capable of modulating the function of tirc7
DE202011103324U1 (de) 2011-07-12 2012-01-02 Nekonal S.A.R.L. Therapeutische anti-TIRC7 Antikörper für die Verwendung in Immun und anderen Krankheiten
WO2018029266A1 (en) 2016-08-09 2018-02-15 Nekonal S.A.R.L. Tirc7 based diagnostic and therapy of cancer
WO2018029296A1 (en) 2016-08-10 2018-02-15 Nekonal S.A.R.L. Tirc7 based diagnostic and therapy of solid cancer
WO2021028579A1 (en) 2019-08-14 2021-02-18 Nekonal S.A.R.L. Method for producing engineered tirc7 specific t-regulatory cells (tregs)

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DK0996726T3 (da) 2004-04-13
AU751151C (en) 2006-09-14
PT996726E (pt) 2004-04-30
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AU9265498A (en) 1999-03-22
DE69820449D1 (de) 2004-01-22
EP1350848A2 (de) 2003-10-08
EP0996726B1 (de) 2003-12-10
AU751151B2 (en) 2002-08-08
CA2301499A1 (en) 1999-03-11
EP0996726A1 (de) 2000-05-03
ATE256185T1 (de) 2003-12-15

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